The distribution of neuroligin4, an autism-related postsynaptic molecule, in the human brain.

NLGN4X was identified as a single causative gene of rare familial nonsyndromic autism for the first time. It encodes the postsynaptic membrane protein Neuroligin4 (NLGN4), the functions and roles of which, however, are not fully understood due to the lack of a closely homologous gene in rodents. It has been confirmed only recently that human NLGN4 is abundantly expressed in the cerebral cortex and is localized mainly to excitatory synapses. However, the detailed histological distribution of NLGN4, which may have important implications regarding the relationships between NLGN4 and autistic phenotypes, has not been clarified. In this study, we raised specific monoclonal and polyclonal antibodies against NLGN4 and examined the distribution of NLGN4 in developing and developed human brains by immunohistochemistry. We found that, in the brain, NLGN4 is expressed almost exclusively in neurons, in which it has a widespread cytoplasmic pattern of distribution. Among various types of neurons with NLGN4 expression, we identified consistently high expression of NLGN4 in hypothalamic oxytocin (OXT)/vasopressin (AVP)-producing cells. Quantitative analyses revealed that the majority of OXT/AVP-producing neurons expressed NLGN4. NLGN4 signals in other large neurons, such as pyramidal cells in the cerebral cortex and hippocampus as well as neurons in the locus coeruleus and the raphe nucleus, were also remarkable, clearly contrasting with no or scarce signals in Purkinje cells. These data suggest that NLGN4 functions in systems involved in intellectual abilities, social abilities, and sleep and wakefulness, impairments of which are commonly seen in autism.

Knockdown of Son, a mouse homologue of the ZTTK syndrome gene, causes neuronal migration defects and dendritic spine abnormalities.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, a rare congenital anomaly syndrome characterized by intellectual disability, brain malformation, facial dysmorphism, musculoskeletal abnormalities, and some visceral malformations is caused by de novo heterozygous mutations of the SON gene. The nuclear protein SON is involved in gene transcription and RNA splicing; however, the roles of SON in neural development remain undetermined. We investigated the effects of Son knockdown on neural development in mice and found that Son knockdown in neural progenitors resulted in defective migration during corticogenesis and reduced spine density on mature cortical neurons. The induction of human wild-type SON expression rescued these neural abnormalities, confirming that the abnormalities were caused by SON insufficiency. We also applied truncated SON proteins encoded by disease-associated mutant SON genes for rescue experiments and found that a truncated SON protein encoded by the most prevalent SON mutant found in ZTTK syndrome rescued the neural abnormalities while another much shorter mutant SON protein did not. These data indicate that SON insufficiency causes neuronal migration defects and dendritic spine abnormalities, which seem neuropathological bases of the neural symptoms of ZTTK syndrome. In addition, the results support that the neural abnormalities in ZTTK syndrome are caused by SON haploinsufficiency independent of the types of mutation that results in functional or dysfunctional proteins.

Identification of novel compound heterozygous mutations in ACO2 in a patient with progressive cerebral and cerebellar atrophy.

BACKGROUND: The tricarboxylic acid (TCA) cycle is a sequence of catabolic reactions within the mitochondrial matrix, and is a central pathway for cellular energy metabolism. Genetic defects affecting the TCA cycle are known to cause severe multisystem disorders. METHODS: We performed whole exome sequencing of genomic DNA of a patient with progressive cerebellar and cerebral atrophy, hypotonia, ataxia, seizure disorder, developmental delay, ophthalmological abnormalities and hearing loss. We also performed biochemical studies using patient fibroblasts. RESULTS: We identified new compound heterozygous mutations (c.1534G > A, p.Asp512Asn and c.1997G > C, p.Gly666Ala) in ACO2, which encodes aconitase 2, a component of the TCA cycle. In patient fibroblasts, the aconitase activity was reduced to 15% of that of the control, and the aconitase 2 level decreased to 36% of that of the control. As such a decrease in aconitase 2 in patient fibroblasts was partially restored by proteasome inhibition, mutant aconitase 2 was suggested to be relatively unstable and rapidly degraded after being synthesized. In addition, the activity of the father-derived variant of aconitase 2 (p.Gly666Ala), which had a mutation near the active center, was 55% of that of wild-type. CONCLUSION: The marked reduction of aconitase activity in patient fibroblasts was due to the combination of decreased aconitase 2 amount and activity due to mutations. Reduced aconitase activity directly suppresses the TCA cycle, resulting in mitochondrial dysfunction, which may lead to symptoms similar to those observed in mitochondrial diseases.

Dopaminergic abnormalities in Hdac6-deficient mice.

Histone deacetylase 6 (Hdac6), a multifunctional cytoplasmic deacetylase, is abundant in brain. We previously demonstrated that global Hdac6 depletion causes aberrant emotional behaviors in mice. Identification of affected brain systems and its molecular basis will lead to new insights into relations between protein acetylation events and psychiatric disorders. Here we report the dopaminergic abnormalities in Hdac6 KO mice. The dopamine transmission mediated by D1-like and D2-like G protein-coupled dopamine receptors is known to play roles in controlling movement, cognition, and motivational processes, and its dysfunction causes psychiatric disorders. We found that Hdac6 KO mice showed significantly increased locomotor response to novel, but not to habituated environment. In addition, Hdac6 KO mice showed a long-lasting sensitivity to psychostimulants, increased locomotor response to D2-like, but not D1 dopamine receptor agonists, and rapid locomotor response to apomorphine, a direct dopamine agonist, in dopamine-depleted condition. Hdac6 protein was expressed in dopaminergic neurons and their terminals in adult mice brain, and Hdac6-depletion augmented acetylation levels of dopamine-enriched synaptosomal proteins. In Hdac6 KO mice, the striatal content of dopamine and its metabolites was normal in basal condition, but mRNA level of D2 dopamine receptor in the striatum was decreased by 30%. Taken together, our results provide evidence that Hdac6 deficiency leads to aberrant dopamine-dependent behaviors by enhancing postsynaptic dopamine D2 receptor response. This study points out the possibility that Hdac6 and reversible-acetylation events play a regulatory role in D2 dopamine receptor signaling, and thus participate in the pathology of the dopamine-related psychiatric disorders such as schizophrenia.

Loss of deacetylation activity of Hdac6 affects emotional behavior in mice.

Acetylation is mediated by acetyltransferases and deacetylases, and occurs not only on histones but also on diverse proteins. Although histone acetylation in chromatin structure and transcription has been well studied, the biological roles of non-histone acetylation remain elusive. Histone deacetylase 6 (Hdac6), a member of the histone deacetylase (HDAC) family, is a unique deacetylase that localizes to cytoplasm and functions in many cellular events by deacetylating non-histone proteins including α-tubulin, Hsp90, and cortactin. Since robust expression of Hdac6 is observed in brain, it would be expected that Hdac6-mediated reversible acetylation plays essential roles in CNS. Here we demonstrate the crucial roles of Hdac6 deacetylase activity in the expression of emotional behavior in mice. We found that Hdac6-deficient mice exhibit hyperactivity, less anxiety, and antidepressant-like behavior in behavioral tests. Moreover, administration of Hdac6-specific inhibitor replicated antidepressant-like behavior in mice. In good agreement with behavioral phenotypes of Hdac6-deficient mice, Hdac6 dominantly localizes to the dorsal and median raphe nuclei, which are involved in emotional behaviors. These findings suggest that HDAC6-mediated reversible acetylation might contribute to maintain proper neuronal activity in serotonergic neurons, and also provide a new therapeutic target for depression.

Consensus substrate sequence for protein-tyrosine phosphatase receptor type Z.

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

Tyrosine phosphorylation of ErbB4 is enhanced by PSD95 and repressed by protein tyrosine phosphatase receptor type Z.

Protein tyrosine phosphatase receptor type Z (Ptprz/PTPzeta/RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) preferentially expressed in the brain. ErbB4 is a member of the ErbB-family tyrosine kinases known as a neuregulin (NRG) receptor. Both are known to bind to postsynaptic density-95 (PSD95) on the second and the first/second PDZ (PSD95/Disc large/zona occludens1) domains, respectively, through the PDZ-binding motif of their carboxyl termini. Here we report a functional interaction between Ptprz and ErbB4. An intracellular carboxyl-terminal region of Ptprz pulled-down PSD95 and ErbB4 from an adult rat synaptosomal preparation. ErbB4 and Ptprz showed co-localization in cell bodies and apical dendrites of neurons in the prefrontal cortex. In HEK293T cells, phosphorylation of ErbB4 was raised by co-expression of PSD95, which was repressed by additional expression of Ptprz. In vitro experiments using the whole intracellular region (ICR) of ErbB4 also showed that PSD95 stimulates the autophosphorylation of ErbB4, and that the ICR of Ptprz dephosphorylates ErbB4 independent of the presence of PSD95. Taken together with the finding that the tyrosine phosphorylation level of ErbB4 was increased in Ptprz-deficient mice, these results suggest that Ptprz has a role in suppressing the autoactivation of ErbB4 by PSD95 at the postsynaptic density in the adult brain.

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases.

Protein tyrosine phosphorylation, controlled by the activities of both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), plays critical roles in a wide variety of cellular events. However, in contrast to the PTKs, our understanding of the biological functions of PTPs has been limited to date. This is mainly the result of the difficulty in identifying the substrate molecules of individual PTPs. We described a genetic method to screen for PTP substrates, which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This system is conceptually applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.

Protein tyrosine phosphatase receptor type Z is inactivated by ligand-induced oligomerization.

Receptor-type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPzeta/RPTPbeta), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G protein-coupled receptor kinase-interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand-induced oligomerization.

Protein tyrosine phosphatase receptor type Z is involved in hippocampus-dependent memory formation through dephosphorylation at Y1105 on p190 RhoGAP.

Ptprz is a receptor-type protein tyrosine phosphatase predominantly expressed in the brain as a chondroitin sulfate proteoglycan. Ptprz-deficient mice exhibit an age (maturation)-dependent impairment of spatial learning in the Morris water maze test and enhancement of long-term potentiation (LTP) in the CA1 region in hippocampal slices. The enhanced LTP is canceled out by pharmacological inhibition of Rho-associated kinase (ROCK), suggesting that the lack of Ptprz causes learning impairment due to aberrant activation of ROCK. Here, we report that Ptprz-deficient mice exhibit impairments in hippocampus-dependent contextual fear memory because of abnormal tyrosine phosphorylation of p190 RhoGAP, a GTPase-activating protein (GAP) for Rho GTPase. We found that phosphorylation at Y1105, a major tyrosine phosphorylation site on p190 RhoGAP, is decreased 1h after the conditioning in the hippocampus of wild-type mice, but not of Ptprz-deficient mice. Pleiotrophin, a ligand for Ptprz, increased tyrosine phosphorylation of p190 RhoGAP in B103 neuroblastoma cells. Furthermore, Ptprz selectively dephosphorylated pY1105 of p190 RhoGAP in vitro, and the tyrosine phosphorylation at Y1105 controls p190 RhoGAP activity in vivo. These results suggest that Ptprz plays a critical role in memory formation by modulating Rho GTPase activity through dephosphorylation at Y1105 on p190 RhoGAP.

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z.

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.

Mice deficient in protein tyrosine phosphatase receptor type Z are resistant to gastric ulcer induction by VacA of Helicobacter pylori.

The vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered intragastrically. Here we report that mice deficient in protein tyrosine phosphatase receptor type Z (Ptprz, also called PTP-zeta or RPTP-beta, encoded by Ptprz) do not show mucosal damage by VacA, although VacA is incorporated into the gastric epithelial cells to the same extent as in wild-type mice. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz-/- mice also showed similar incorporation of VacA, cellular vacuolation and reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane 24 h after treatment with VacA. VacA bound to Ptprz, and the levels of tyrosine phosphorylation of the G protein-coupled receptor kinase-interactor 1 (Git1), a Ptprz substrate, were higher after treatment with VacA, indicating that VacA behaves as a ligand for Ptprz. Furthermore, pleiotrophin (PTN), an endogenous ligand of Ptprz, also induced gastritis specifically in Ptprz+/+ mice when administered orally. Taken together, these data indicate that erroneous Ptprz signaling induces gastric ulcers.

Genetic labelling of specific axonal pathways in the mouse central nervous system.

We report on transgenic mouse lines in which several sensory systems in the brain are specifically visualized genetically. We employed GAP-lacZ as an axon-targeted reporter protein that was constructed by fusing the membrane-anchoring domain of the GAP-43 protein to lacZ. The reporter gene was introduced into the genome under the control of a promoter element of Brn3b transcription factor to establish transgenic mouse lines. The individual lines thus generated afforded clear images of specific axonal pathways of the visual, vomeronasal, pontocerebellar, and auditory systems. The reporter protein labelled the entire axonal process as well as the cell body of developing and mature neurons on staining with X-gal. We show that these lines facilitate the developmental and anatomical study of these neural systems. This strategy should be applicable to a variety of neural systems by using various specific promoter elements.